PT  - JOURNAL ARTICLE
AU  - Spencer Lopp
AU  - Robin W. Yeo
AU  - Nishant Jha
AU  - Xiao Yang
AU  - Gabriella Alvarez
AU  - Courtney Klappenbach
AU  - Daniel O. Hart
AU  - Lei S. Qi
AU  - Timothy P. Daley
TI  - casmini-tool: a comprehensive database for efficient and specific guide RNA design using dCasMINI
AID  - 10.1101/2023.09.17.558168
DP  - 2023 Jan 01
TA  - bioRxiv
PG  - 2023.09.17.558168
4099  - http://biorxiv.org/content/early/2023/09/18/2023.09.17.558168.short
4100  - http://biorxiv.org/content/early/2023/09/18/2023.09.17.558168.full
AB  - The dCasMINI protein is a hyper-compact, nuclease-inactivated CRISPR-Cas system engineered for transcriptional modulation and epigenetic editing [Xu et al., 2021]. The small size of dCas-Mini (529 amino acids), less than half the size of comparable Cas9 molecules, makes it ideal for AAV-based therapies which are frequently limited by AAV’s small cargo capacity. Unlike Cas9 or Cas12a, there is no available computational tools for designing CasMINI guides. To facilitate and accelerate the development of dCasMINI-based applications, we synthesized knowledge regarding CasMINI guide design and built a website to assist researchers in designing optimal guides for dCasMINI-based experiments for transcriptional inhibition (CRISPRi) and activation (CRISPRa), which covers 99.7% of genes for CRISPRi and 99.9% of genes for CRISPRa. We experimentally characterized the importance of each nucleotide position on the guide RNA for determining its activity. Based on this information,, our tool offers more sensitively mapping off-targets and provides information about alignment mismatches in the spacer seed region, which we have experimentally determined to be critical for true binding events. The tool is freely available at casmini-tool.com.Competing Interest StatementL.S.Q. is the founder of Epicrispr Biotechnologies, and also serves as a scientific advisor for Laboratory of Genomics Research and Kytopen. S.L., R.W.Y., X.Y., D.O.H., L.S.Q., and T.P.D. hold provisional patents relating to this work, are employees of and acknowledge outside interest in Epicrispr Biotechnologies.